PROJECT SUMMARY Uterine leiomyoma (LM) is a benign smooth muscle tumor of the myometrium with an overall prevalence of over 70%. Symptoms of this tumor include heavy menstrual bleeding, pelvic pressure or pain, and often require intervention. Since there is no effective non-surgical therapy for LMs, approximately a third of women with a diagnosis of LM undergo hysterectomy, which imposes a significant physical and psychological burden. Surgery also creates serious financial strain, with the annual cost for surgical management of LMs in the United States estimated to be 829 million to 4.3 billion dollars. Hence, novel therapeutic options with long-term efficacy to replace surgical management are urgently needed. In order to develop such medical options, a better understanding of the cellular and molecular pathogenesis of LM is essential. Some studies suggest that stem cells play a critical role in the pathogenesis, yet the identity and function of LM stem cells (LM-SCs) remain ambiguous as different groups propose several incompatible stem cell models. This confusion is partially due to faulty presumptions that all LMs are equivalent, and that all cells in a LM arise from a single progenitor cell. Accumulating evidence indicates that LMs contain substantial non-tumor cells of independent cell lineages, and the composition of tumor and non-tumor cells is distinctive between different LM subtypes. In this project, we will focus on the MED12 mutant LM (MED12-LM), the most prevalent LM subtype accounting for ~70% of all LMs. In MED12-LM, causal MED12 mutations are present only in tumor smooth muscle cell (T-SMC), which accounts for ~60 % of total cell population. Tumor-associated fibroblast (TAF), which is negative for MED12 mutations, is the second major cell type in MED12-LM, accounting for ~40% of total cells. Surprisingly, previous studies that proposed LM-SC models did not recognize the presence of TAFs. In our analysis, the putative LM-SC fractions primarily consisted of non-tumor cells. Accordingly, we seek to determine presence and characteristics of LM- SC in MED12-LM utilizing the patient derived xenograft (PDX), the only research model that can replicate the hormone-dependent growth of MED12-LMs. To identify LM-SCs, we will identify cell types that compose MED12- LM by single cell RNA-seq (scRNA-seq) of PDXs. By comparing resting (no hormone) and growing (with hormones) PDXs, we will identify cells and genes that control hormone-dependent growth of MED12-LMs. We will also determine the growth kinetics of each cell type during regeneration of PDXs by a Pulse-Chase assay with three thymidine analogs. Due to their infrequent proliferation, tissue stem cells are often identified as label- retaining cells (LRCs). By this assay, we will identify LRCs as well as the first cells that undergo cell division in response to hormones. Integration of scRNA-seq and Pulse-Chase assays will reveal the identity of cells and signaling pathways that are essential for MED12-LM growth. Such cells/pathways can be targets of novel preventive and therapeutic treatments for this highly prevalent tumor.